Particles, method of preparing said particles and uses thereof

ABSTRACT

Particles, especially colloidal particles, comprising an interior phase of a non-lamellar reversed cubic, intermediate or hexagonal liquid crystalline phase, or a homogeneous L3 phase, and a surface phase of a lamellar crystalline or liquid crystalline phase, or an L3 phase. A method of preparing such particles by creating a local dispersible phase, within the homogeneous phase, preferably by means of a fragmentation agent, and fragmentating the homogeneous phase so as to form said surface phase. Several medical as well as non-medical uses of the particles referred to, e.g. as an antigen-presenting system, as a delivery system for anticancer, antifungal and antimicrobial drugs, and as carriers of nucleic acids or nucleotides.

This is a application of PCT/SE92/00692 filed under 35 USC 371, which isa continuation of U.S. application Ser. No. 07/771,014 now abandoned.

1. FIELD OF THE INVENTION

The present invention relates to the field of amphiphilic-solvent basedsystems and more specifically To the fragmentation of such systems bymeans of a novel method. Thus, by means of said novel method systems,which are otherwise homogeneous and in equilibrum, can be fragmentedinto small particles, especially colloidal particles. These newparticles are extremely well suited as e.g. drug delivery particles orsystems, but are also useful for several other medical as well asnon-medical applications. Thus, the invention also encompasses said newparticles as well as valuable uses thereof.

2. BACKGROUND OF THE INVENTION

The use of reversed cubic and liquid crystalline phases in the field ofcontrolled release devices is described in EPO 125 751 (Engstrom et al1983). However, there are many applications where the use of ahomogeneous phase is not manageable. By the present invention it hasbecome possible to prepare particles, especially colloidal particles, ofsuch phases or similar phases, viz by means of a new fragmentationtechnique. The importance of said new technique, and the new particlesobtained thereby, can be better understood from the following briefreview of the prior art concerning other Types of particles andhomogeneous phases.

2.1. Dispersed lipid-based systems in pharmaceutical preparations

Essentially, there have To date been three major particulate colloidallipid-water systems which have been considered as suitable for drugdelivery, namely such based on the lamellar mesophase as liposomes,micellar-based phases including micelles, reversed micelles, and mixedmicelles and various kinds of emulsions including microemulsions, aswell as more novel carriers as ISCOM's (Morein 1988) (a general textconcerning these systems is Pharmaceutical dosage forms, Dispersesystems 1988). The latter system has been utilized for intravenousnutrition since the beginning of this century and as an adjuvant systemknown as the Freunds adjuvant. These are of oil-in-water (O/W) andwater-in-oil (W/O) types, respectively. Liposomes have since theirdiscovery been extensively investigated as drug delivery systems forvarious routes and drugs. The development of new colloidal drug carriersystems is a research area of intensive activity and it is likely thatnew systems, especially new emulsion based systems, will appear in thenear future (cf. Weiner 1990a, 1990b). Lipid-based vehicles can takeseveral different morphological forms such as normal and reversedmicelles, microemulsions, liposomes including variants as unilamellar,multilamellar, etc., emulsions including various types as oil-in-water,water-in-oil, multiple emulsions, etc., suspensions, and solidcrystalline. In addition so called niosomes formed from nonionicsurfactants have been investigated as a drug vehicle. The use of thesevehicles in the field of drug delivery and biotechnology is welldocumented (Mulley 1974, Davis et al. 1983, Gregoriadis 1988a,Liebermann et al 1989). Particularly in the field of drug delivery theuse of lipid-based drug delivery systems, especially dispersed systems,has attained increasing interest as the pharmaceutical industry isdeveloping more potent and specific--and thus more (cyto)toxic--drugs.This is because the vehicle can in principle reduce such toxic effectsand/or side-effects, due to sustained release or increasedsite-specificity. The current invention is easily distinguished fromthese earlier lipid-water based systems, as follows:

The term liposomes is conceptually wrong in view of the currentknowledge of polymorphism of lipids. Liposome means "lipid body" and hasby many authorities in the field been defined as any structure with anenclosed volume that is composed of lipid bilayers (see eg. Tice andTabibi 1992). This is not only very misleading but also conceptuallywrong. Such a definition means that any dispersed lipid based structurebuilt up by a bilayer should fall into this category of device withoutregarding the different crystallographic aspect of the undispersed,homogeneous, phase from which the particulate vehicle is derived. Itwould, however, not include dispersions in which the interior of theparticles is made up by reversed hexagonal phases, since they are builtup by a monolayer, rather than a bilayer. Unfortunately, the concepts oflipid polymorphism and in particular the more complex structures ofcubic liquid crystalline phases are often overlooked. Since the currentdisclosure is in the field of lipid-based vehicles, in which variousreversed lyotropic liquid crystalline phases are enclosed in a volumewhose boundary is made up by L3 phase or lamellar crystalline phase orlamellar liquid crystalline phase, or a combination thereof, it shouldbe stressed that the current invention encloses either lipid bilayer ormonolayer structures different from the lamellar phase. The orderedinterior of each particle in the current invention is a portion of alipid-water microstructured phase that is a thermodynamically stablephase, either a cubic, hexagonal, intermediate phase or an L3 phase. TheL3 phase is not classified as a liquid crystalline phase, as the others,rather it is an isotropic solution phase, using the standardnomenclature in the literature of amphiphile microstructures. Thephysical properties of the homogeneous reversed liquid crystallinephases used in the currect disclosure are those presented in the patentby Engstrom et al. (1983) referred to. In the cases where a cubic phaseconstitutes the interior of the particles it is built up by a bi- ormulticontinuous interpenetrating network microstructure, at the scale ofnanometers. This makes these phases unique with regard tocompartmentalization since the two independent interpenetrating networksseparated by the bilayer can be distinguished, and endows them withextremely high specific surface area, which is especially important inthe formulation of amphiphilic drugs straddle hydrophobic andhydrophilic microdomains.

The current invention is thus easily and sharply distinguished from bothliposomes, emulsions, microemulsions, as well as variousmicroencapsulated emulsions, hydrogels, and reversed micelles. Mostobviously, the interior phase(s) of the current particles is (are) athermodynamic equilibrium phase, and thus appears as a discrete regionin a phase diagram which obeys the phase rule of Gibb's and other lawsof chemical and thermodynamical equilibria; this is in sharp contrastwith liposomes and emulsions, which are non-equilibrium states ormorphologies. [Note that we are using the convention of referring toequilibrium structures as "phases" and non-equilibrium structures as"states"]. In the case of emulsions the interior is also thermodynamicalstable, but it is an interior which lacks long-range order, and is notcomposed of either lipid bilayers or lipid monolayers, or analogousstructural elements. This is a clear distinction, which is directlyaccessible to experiment, since the interior phase used in the currentparticles give rise to Bragg peaks on examination with small-angle X-ray(or neutron) scattering techniques, in accordance with its latticeordering; thus the Bragg peaks recorded can be indexed to e.g. a simplecubic, body-centered cubic, or face-centered cubic lattice, hexagonallattice, or tetragonal lattice in the case the interior is made up by acubic phase, a hexagonal phase or intermediate tetragonal phase,respectively. In contrast, no case has ever been reported in whichmultiple Bragg peaks, indexing to any of these lattices, were recordedin a small-angle scattering experiment on a liposomal dispersion or anemulsion. Clearly, the surface of the current particles can in practicegive rise to diffraction indexing on a lamellar lattice. In the case ofmicroemulsions and reversed micellar phase, both lacking long rangeorder, they are clearly distinguished from the L3 phases in the currentsurfactant literature.

The distinctions between the current invention on the one hand, and theliposomal dispersions and emulsions on the other, then follow directlyfrom the above distinctions, and it is only in the case of the reversedliquid crystalline phase dispersions disclosed herein the interiors ofthe particles are substantially composed of regions of reversed liquidcrystalline phase(s).

In the case of dispersions of L3 phases, the interiors of the particlesare not composed of liquid crystalline material but of the to the cubicphases closely related L3 phases. The L3 phases are thermodynamicequilibrium phases, distinguishing them from liposomes and emulsions asin the case of cubic phases. The lipid film forms a highly connectedbilayer as in the cubic phase, again in contrast with the liposomes andemulsions. However, in this case scattering experiments do not reveallong-range order as in the cubic phase.

Of special importance in formulations, used either for drug delivery orfor biological or biotechnological applications, is the position andorientation of the compound with respect to-the bilayer. In the currentinvention specific orientation may be readily achieved in the case theinterior is composed of cubic phase, for which it is an inherentproperty, as opposed to liposomal bilyaers. This substantiallysimplifies the process of standardizing enzyme activity in theformulation. Such selectivity in membrane topography is not easilyestablished in other lipid-based systems such as liposomes andemulsions. There are several other areas of interest where the presentedtopography of the compound is of profound importance, as with antigenpresentation in immunization processes. The current invention canaccomplish the optimization of this presentation for both extracellularand intracellular targets.

2.2. Homogeneous liquid crystalline phases in pharmaceuticalpreparations

Liquid crystals do participate in the microstructure of pharmaceuticalpreparations, and probably do so more frequently than is usuallyexpected. The use of homogeneous reversed cubic and hexagonal phases asa controlled release system for use in e.g. drug delivery systems wasinvented by Engstrom, Larsson, and Lindman in Lund, Sweden, who areholders of a current patent (Engstrom et al. 1983, see also Ericsson etal 1991, and references therein). Dr. D. Attwood and coworkers inManchester, UK, have also investigated the use of cubic phases for thepurpose of drug delivery (cf. Burrows et al 1990).

Cf. also Mueller-Goymann and collaborators (Mueller-Goymann 1985,Mueller-Goymann 1987, Mueller-Goymann 1989 and references within theseworks). Other contributions occur in the literature (cf. Ibrahim 1989,Tyle 1990) and are not restricted to lyotropic liquid crystals (Loth andEuschen 1990).

2.3. Dispersed reversed cubic liquid crystalline phases

There have been speculations of the existance of dispersed cubic liquidcrystalline phases in connection with fat digestion (cf. Lindstrom et al1981) and recently Larsson (1989) suggested a structure of such cubicphase dispersions; in these, the surface layer was proposed to be alamellar phase, which immediately distinguishes such dispersions fromthe particles whose surface phase is L3 phase disclosed herein--theparticles in this embodiment of the present invention are isotropicthroughout, whereas those discussed by Larsson (1989) containanisotropic, birefringent regions which are easily detected in thepolarizing microscope. The only exception is particles, described in thepresent disclosure, which are surrounded by a lipid structure which iscrystalline, not liquid crystalline as in the lamellar phase. Regardingthe case of dispersions of reversed liquid crystalline phases by the useof a lamellar liquid crystalline phase as a dispersable phase the novelfragmentation technique according to the invention can be used.

2.4. Phase behavior in lipid-water based systems and the determinationof cubic phases

A "lipid" is, in a broad view, defined as any molecule containing asubstantial part of hydrocarbon. However, only those lipids that containa hydrophilic polar part can give rise to liquid crystals byinteractions with water. The basis for lipid lyotropic (andthermotropic) mesomorphism, and the formation of lipid assemblies, isthe duality in solubility resulting from the presence of apolar(hydrophobic) and polar (generally hydrophilic) regions of thesurfactant molecule--that is, its amphiphilicity (or amphipaticity).Amphiphilic lipids can be classified according to their interactionswith water into nonpolar and polar (Small 1986). Where applicable withinthis disclosure we are concerned with lipids or lipid-like amphiphilesthat exhibit mesomorphism and are thus classified as polar, insolubleand swelling amphiphiles. If nothing else is said we use the terminologyintroduced by Luzzati and associates (see Mariani et al. 1988, and ref.therein).

The principal techniques for studying the different phase structures arepolarizing microscopy, X-ray diffraction, nuclear magnetic resonance(NMR) spectroscopy and electron microscopy techniques. Other techniques,as differential scanning calorimetry (DSC) and rheology can be used togive complementary information. Unambiguously phase determinations ofthe phases constituting the interior as well as the exterior is aprerequisite in order to classify dispersions according to the currentinvention. Preliminary phase behavior is usually carried out by textureanalysis between crossed polarizers and more detailed in a polarizingmicroscope (Rosevear 1968). X-ray diffraction techniques are the obviousmethods to deduce the symmetry of liquid crystals. The characterizationof lipid mesophases by diffraction (Luzzati 1968) is based firstly onsymmetry and the interpretation is normally based on treating thediffraction photographs as powder patterns. The long-range order of theassemblies in either one, two, or three dimensions, give rise toreflections which are converted to interplanar spacings. It is only withX-ray diffraction studies phase assignments can be regarded asunambiguous.

2.4.1. Phase diagrams in lipid-water systems

Fontell (1990) gives a comprehensive and systematic reveiw on cubicphase forming lipids and lipid-like surfactants and the occurrence ofthe cubic phases in the phase diagram and their relation to otherphases. The information obtained from the structure of the neighboringphases can often be valuable for the identification of a cubic phase.The fact that a mesophase, such as the cubic or hexagonal phase, is inequilibrium with excess of water, is itself a strong indication that thestructure is of the reversed, type II topology.

In the context of this invention, two examples of lipid-water basedsystems have been investigated with the objective of mapping theunderlying phase behavior so as to understand and develop the techniquesdisclosed herein regarding the fragmentation process: Commerciallyavailable products have been used throughout this study, and it isimportant to note that these are generally not single-componentproducts. We first discuss the binary phase diagram of the glycerolmonooleate (GMO)-water system. The GMO has been obtained throughmolecular distillation of pine-needle oil (Grinsted, Denmark), and has amonoglyceride content of >98%, of which 92.3% is monoolein (MO) (MOrefers to the pure monoolein, while GMO refers to a monoolein richmonoglyceride blend). Many phase diagrams have been reported involvingcubic phases of monoglycerides (Lutton (1965), Larsson et al. 1978, Krogand Larsson 1983, Larsson 1989, Krog 1990). In addition to the purelipids monoolein, monoelaidin, monolinolein, monoarachidin, andmonolinolein (Lutton 1965, Larsson et al. 1978, Hyde et al. 1984,Caffrey 1989), several blend qualities of monoacylglycerides are wellcharacterized and known to form cubic phases in equilibrium with water(Larsson and Krog 1983, Krog 1990). Significantly, these blends areavailable at low production costs, typically less than $2 per pound.

Monoacylglycerides are often used in cosmetic products (CosmeticIngredient Review expert panel 1986), food industry (Krogh 1990) andpharmaceutics (Martindale the extra pharmacopoeia 1982), and aregenerally recognized as safe (GRAS) substances and as indirect foodadditives for human consumption without restrictions as to theirconcentrations. Federal regulations allow the use of monoglycerides,blends thereof, and blends of mono- and diglycerides as prior-sanctionedfood ingredients and as both indirect and direct food additives.Furthermore, the metabolic fate of monoglycerides (and glycerides ingeneral) is well documented in the human body. In the cosmetic industrymonoglycerides and blends thereof, especially monoolein, are used asemulsifiers and thickening agents and recognized as safe cosmeticingredients at concentrations up to 5% (Cosmetic Ingredient Reviewexpert panel 1986).

The fact that there exists cubic phases in equilibrium with excess ofwater in the above mentioned monoglyceride systems is a strongindication that the cubic phase is of the reversed, type II topology.This has been verified by self-diffusion NMR (Lindblom et al. 1979). Itshould be pointed out that several systems which form cubic phases ofthe reversed type exhibit cubic mesomorphism, i.e. the appearance of asequence of distinguishable cubic phases with different physicalappearance, as well as exhibiting different lattice characteristics. Thephase behavior of the present GMO-water system was; found to be verysimilar to that of MO-water reported by Hyde et al. (1984) (Engstrom andEngstrom 1992). The Q²²⁴ was found to be the cubic phase which coexistswith excess of water.

The second lipid-water system used is the ternary system of GMO-soybeanlecithin (SPC)-water. SPC is a pure phosphatidylcholine with the tradename Epikuron 200 which is well-characterized (Bergenstahl and Fontell1983). It shares the general features of the phase diagram forMO-dioleoyl phosphatidylcholine-heavy water system reported by Gutman etal (1984). The existence of three cubic phases within the cubic regionis experimentally verified by X-ray diffraction, as was the coexistenceof cubic phases with excess of water.

2.4.2. Phase behavior and phase diagrams in lipid-protein-water systems

The phase properties in lipid-protein-water mixtures is a relativelyunexplored area of research. Most of the studies have been reported bythe Groups of Gulik-Krzywicki, Luzzati and colleagues (cf. Mariani etal. 1988, Gulik-Krzywicki 1975), by De Kruijff and coworkers (cf.Killian and De Kruijff 1986) and by Larsson and coworkers (cf. Ericssonet al. 1983, Ericsson 1986). Most studies address the behavior indiluted systems and often deal with the stability of the lamellar phasevs. the reversed hexagonal phase. The induction of non-lamellar phasesis well established-for quite many systems. Some works address the phaseproperties vs. the activity of membrane bound enzymes, and it has beenpossible in some works to establish a correlation between an increasedenzyme activity and isotropic movement of the lipid matrix. In the fieldof enzyme catalysis in microemulsions, some studies deal with the phasebehavior; however, few works present phase diagrams.

That the cubic phases in the monoolein (MO)-water system could hostquite large amounts of various substances, included proteins, had beenknown for many years (cf. Lindblom et al. 1979). The phase diagram ofMO-lysozyme-water displays the general features of MO-protein-watersystems, in cases where the protein is located in the aqueous labyrinthsof the cubic phase (Ericsson et al. 1983). Ericsson (1986) reported aconsiderable number of proteins which can be incorporated within theMO-water cubic phase.

The second system which has been investigates in considerable detail isthe MO-cytochrome c-water system reported by Luzzati and coworkers(Mariani et al. 1988), and it exhibits the general features found in theMO-lysozyme-water system. However, it also shows some features whichnecessary must arise from the protein; noteworthy is the existence of achiral, non-centrosymmetric cubic phase, with space group 212. Theseaqueous MO-protein systems all exhibit at least one cubic phase whichfulfils the criteria for constituting the interior phase of theparticles according to the present invention.

2.5 Structure of the interior phases

The interior of the particles according to the invention consists ofreversed lyotropic liquid crystalline phases, chosen from the group ofreversed cubic liquid crystalline phases, reversed intermediate liquidcrystalline phase, and reversed hexagonal liquid crystalline phase, orL3 phase, or a combination thereof. These phases are all wellcharacterized and well established in the field of polymorphism oflipids and surfactants.

2.5.1. Structure of the cubic and hexagonal phases

Several reviews are available where cubic phases are discussed; see e.g.Luzzati (1968), Fontell (1974, 1978, 1981), Ekwall (1975), Tiddy (1980)and Luzzati et al. (1986). In recent years several surveys devoted tocubic phases have appeared. Luzzati and associates (Mariani et al. 1988)(see also Luzzati et al. 1987) give a detailed crystallographicdescription of the current situation with regard to the structure of thesix cubic phases observed so far. Lindblom and Rilfors (1988) havereviewed the occurrence and biological implications of cubic phasesformed by membrane lipids, and Larsson (1989) has reviewed the latestdevelopments in the study of cubic lipid-water phases. A comprehensivereview of the occurrence of cubic phases in literature phase diagramswas recently presented by Fontell (1990).

A general classification of the cubic phases is still not available.However, in the case of bilayer-bicontinuous cubic phases in binarysystems they can be classified according to their interfacial meancurvature as "normal" (type I) or reversed (type II) cubic phases. Typecubic phases are those whose mean curvature at the apolar/polarinterface is toward the apolar regions. Contrarily, type II or reversedcubic phases are those whose interface is towards the polar regions. Inconnection with the invention we are only concerned with cubic phases oftype II, i.e. reversed.

Regarding the structure of the hexagonal phase it consists ofhexagonally arranged rods of water (solvent) surrounded by a monolayerof amphiphile (see e.g. Seddon 1990, for a review).

2.5.2. Structure of the L3 phase

The microstructures of the L3 phases referred to are similar to thosefrequently found in surfactant-water systems (Benton et al. 1983, Porteet al. 1988, Gazeau et al. 1989, Anderson et al. 1989, Strey et al.1990a, Strey et al. 1990b, Milner et al. 1990). The acquiescent L3 phaseis isotropic. However, one striking and characteristic feature is thatit shows extended flow birefringence. Other characteristics include longequilibration times and, at least relative to the amphiphileconcentration, high viscosity. The structure is generally believed to bebuilt up of multiply-connected bilayer forming a bicontinuous structureof high connectivity, and it may be regarded as a disordered counterpartto the cubic phases (Anderson et al. 1989), possessing similartopological connectivity and a local bilayer structure, but lackinglong-range order.

2.6. Structure of the surface or dispersable phases

The structure of the L3 phase when used as the dispersable orfragmenting phase is exactly as described in 2.5.2. It should be pointedout that one bilayer of an L3 phase can not readily be distinguishedfrom a lamella of a diluted lamellar phase. Similarly, it has beenpointed out that the L3 phase may in certain systems exhibitmetastability (Dubois and Zemb 1991) in which a transformation of the L3phase to a lamellar phase was observed after 3 weeks of equilibrationtime. The lamellar structures, including lamellar phases with:disordered chains, untilted ordered or gel, and tilted gel, used as thedispersable phases are described by Luzzati (1968).

3. DISCLOSURE OF THE INVENTION

The present invention relates to new particles, especially colloidalparticles, made from reversed cubic, hexagonal or intermediate phases,or L3 phases, or mixtures thereof, by fragmentation of the correspondinghomogeneous structure. Fragmentation can be achieved through severalprocesses described below. The resulting particles are thus composed ofan interior amphiphilic-based phase surrounded by a surface phaseanchored to the bi- or mono-layer of the interior phase. The propertiesof the surface phase is such that it can easily be dispersed.

Thus, the present invention is in the field of lipid-based dispersedvehicles representing novel drug delivery systems. The invention isnonetheless sharply distinguished from liposomes and emulsions, andsimilar particulate vehicles as well as from the techniques used for thepreparation of such lipid-based particulate systems. The class ofdelivery vehicles claimed comprises particles whose interiors aresubstantially composed of lyotropic liquid crystalline phases of bilayeror monolayer type, or the closely related L3 phases which lacklong-range super-molecular order; the reversed lyotropic liquidcrystalline phases can be chosen from the group consisting of The cubicphase, the hexagonal phase, and the intermediate phase, or a combinationthereof, using the nomenclature in the current surfactant literature.These liquid crystals are thermodynamic equilibrium phases, in contrastwith liposomes and emulsions which are metastable. With the interior ofThe current particles being liquid crystals, they exhibit Bragg peaks insmall-angle X-ray scattering (SAXS) experiments, as opposed to liposomesand emulsions which do not possess long-range crystallographic order onthe microstructural length scale, namely lattice parameters in the rangeof nanometers or more. Dispersions of the liquid crystals loaded with anactive compound can be conveniently prepared by fragmentation of thehomogeneous liquid crystal. A variety of techniquies disclosed hereincan be used for The fragmentation process, creating different surfaceproperties of the particles, depending on the choice of dispersablephase and its composition. The fragmentation can be spontaneous or aidedby standard homogenizing means such as valve homogenizers. Thedispersions can display long-term stability.

More specifically The new particles according to the invention comprisean interior phase of a non-lamellar lyotropic liquid crystalline phaseselected from the group consisting of a reversed cubic liquidcrystalline phase, a reversed intermediate liquid crystalline phase anda reversed hexagonal liquid crystalline phase, or a homogeneous L3phase, or any combination thereof, and a surface phase selected from thegroup consisting of a lamellar crystalline phase and a lamellar liquidcrystalline phase, or an L3 phase, or any combination thereof.

Thus, the invention makes use of non-lamellar, but equilibrium, reversedlyotropic liquid crystalline phases that occur in many lipid-water andother amphiphile-solvent based systems. The following terminology isused: The particles whose inner is made up by non-lammelar phases, theinterior phase, are prepared by a novel fragmentation procedure whichmakes use of the introduction of disclinations/defects in the interiorphase by the local formation of a dispersable phase such as the L3phase, lamellar liquid crystalline phase, or lamellar crystalline phase,or a combination thereof. The so formed disclinations, whose boundariesmake up the dispersable phase referred to as the surface phase of theparticles, in turn constitute the boundary of a fragment of the interiorphase of the particles. The fragmentation procedure takes place in sucha way that it guarantees the coexistence of the phase making up theinterior, the phase making up the surface, and the solvent-rich solutionphase. The latter is most often rich in water, or any other polarsolvent, or solvent in which the interior phase(s) of the particles is(are) formed. A three phase region can hence be determined as the regionof which these phases coexist and in which the interior phase isfragmented according to above. The particle size can thus be varied to acertain extent since the amount of dispersable phase will determine themaximum sum of surface area of the particles. The invention makes use ofphases constituting the interior phase chosen from the group of reversedcubic liquid crystalline phases, reversed hexagonal liquid crystallinephase, and reversed intermediate liquid crystalline phase, or acombination thereof, or an L3 phase. It is in fact a prerequisite thatwhen the interior phase is a liquid crystalline phase it is of reversedtype since it must be able to coexist with the solvent-rich phase.

More specifically the method according to the invention comprisesforming a homogeneous, non-lamellar lyotropic liquid crystalline phaseselected from the group consisting of a reversed cubic liquidcrystalline phase, a reversed intermediate liquid crystalline phase anda reversed hexagonal liquid crystalline phase, or a homogeneous L3phase, or any combination thereof, creating a local dispersible phase,within said homogeneous phase, of a phase selected from the groupconsisting of a lamellar crystalline phase and a lamellar liquidcrystalline phase, or an L3 phase, or any combination thereof, in thepresence of a solvent phase, said solvent being of a nature with whichsaid homogeneous phase can coexist and wherein said dispersible phasecan be dispersed, and fragmentating said homogeneous phase so as to formparticles, the interior phase of which comprises said homogeneous phaseand the surface phase of which comprises said dispersible phase.

Generally, a fragmentation agent is used to establish the finalappearance of the interior phase as well as the surface phase, eventhough it may only be a change in lattice parameter of the interiorphase, or the establishment of a new interior phase not present in thesystem lacking the fragmentation agent.

The structure of the surface phase can vary depending on the preparationof the particles to be either diluted lamellae (lamellar liquidcrystalline phase), lamellar crystalline phase, or an L3 phase. Thecolloidal fragmented L3 phase particles are made from cubic phasesthrough lyotropic phase transformation of the dispersed cubic phase.Alternative formulations resulting in substantially the same finalmicrostructure for the dispersion fall within the scope of thisinvention.

Especially preferable embodiments of the particles as well as the methodaccording to the invention show those characteristic features which areclaimed in the accompanying claims. These embodiments as well as otherembodiments of the invention will now be described more in detail.

3.1. Dispersions of reversed liquid crystalline phases

A convenient starting point for the formation of cubic phase dispersionsis a cubic phase that can be in thermodynamic equilibrium with excess ofwater or aqueous (molecular or dilute micellar-like) solution. Wedescribe only the invention exemplified in detail with the case of cubicphase interior, since exactly the same procedure can be applied tosytems possessing a reversed hexagonal or intermediate phase in excessof water. Several systems in which a cubic phase coexists with a verydiluted ,aqueous solution have been described in the literature (for areview, see Fontell 1990), all of which can in principle be applied tothe current invention. Many monoacylglyceride-water systems possess thisfeature (see 2.4.) and are suitable to exemplify the invention.

MO may be considered as a fusogenic lipid and can generally not beregarded as blood compatible, at least not as a monomer or as assembledin the cubic phase. However, the particles claimed are blood compatible(with the exception of the dispersion with a crystalline outer palisade,discussed below) as indicated by the lack of lysis products afterincubation with red blood cells for 1 hr. This may be attributed to thevery hydrophilic palisade layer constituted by the surface phase,surrounding the particles. The surface phase can conveniently be chosento be composed of polyethylenoxide units or glyco- moleties, or amixture of these. In these cases, the palisade to some extent mimics theglycocalyx of blood cells. The chemical constituents of the cubic phasecan further be varied by exchanging the monoglycerides by phospholipidssuch as soybean lecithin, egg yolk lecithin, pure phospholipids asdioleoylphosphatidylcholine, and diglycerides. By such means, themolecular constituents of the bilayer structure can be systematicallyvaried so as to achieve the desired properties as described in detailbelow.

Solubilized (active) component(s) also play a role in the finalproperties of the formulation, especially if large amounts of activesubstance are incorporated. To date, every compound which has beensolubilized in the aqueous networks of a reversed cubic phase has beenfound to increase in solubility in comparison with that in aqueoussolution. This has been found to be particularly pronounced in caseswhere the amphiphilic character of the compound calls for the unique,amphiphilic compartmentalization afforded by the cubic phase, so thatboth solubility and stability are increased in the cubic phase. Suchcompounds are e.g. global proteins and glycoproteins, polynucleotidesand highly reactive lipids as prostaglandins and their derivatives asthromboxanes. We strongly emphasize that the current invention isapplicable to any reversed liquid crystalline phase defined as theinterior, and to any of the defined surface phases, and theircoexistence in any Type of solvent-rich media (solution phase)regardless of their molecular composition. The formulation of differentmolecules in the reversed liquid crystalline cubic and hexagonal phasesis described by Engstrom et al. (1983).

Reversed cubic liquid crystalline phases can generally be fragmented byone of the following procedures a)-d):

a) Add, to the equilibrated homogeneous cubic or intermediate phase, anaqueous solution, not necessarily any molecular solution, of one or moreamphiphilic block copolymers where the hydrophilic lipophilic balance(HLB) of the block copolymer is higher than 15. Subsequent stirring withthe appropriate equipment results in a coarse dispersion of fragmentedcubic phase which can be aftertreated as described below. Fragmentationagents belonging to this group are certain poloxamers, such as poloxamer407 and poloxamer 188 (Lundsted and Schmolka 1976a, Lundsted andSchmolka 1976b, Schmolka 1969) and certain amphiphilic proteins, such ascasein.

Examples of other surface active polymers that can potentially be usedas fragmentation or stabilization agents, either alone or in mixtureswith the above, are glycoproteins as mucins and polysaccharides asalginate, propylene glycol alginate, gum arabic, xanthan, carragenan,polyvinylpyrrolidone (PVP) and carboxymethyl-cellulose.

b) Add, to the equilibrated homogeneous reversed liquid crystallinephase, an aqueous dispersion of a mixture of one or more amphiphilicblock copolymers, such as in procedure b), and lipids, such asphospholipids, preferably such mixtures of phospholipids. The ratio oflipid to polymer should not be greater than required to maintain orestablish the liquid crystalline phase constituting the interioraccording to above.

c) Fragment the homogeneous cubic phase by means of ultrasonic devicesunder controlled conditions in a solution of amphiphilic substance(s),again generally of HLB 15 or more. Procedure c) can also be used inconjunction with procedures a) and b) to shorten processing times.

d) Co-equilibrate the starting material, at elevated temperature, withan amphiphilic substance that forms a cubic or intermediate phase at theequilibration temperature and one of the following structures at thetemperature desired for the formulation (typically 37° C., physiologictemperature): 1) a lamellar structure; 2) a lamellar crystallinestructure; 3) an L3 phase. The fragmentation procedure is brought aboutthrough rapid cooling of the system in which one of the structures 1-3is formed at well-defined crystallographic planes in the cubic phase orat defects in the cubic phase. Examples of substances which can bepotentially used for the introduction of particular surface phasesinclude: class 1) phosphatidylcholines such as phosphatidylethanolamineand ester derivatives thereof, phosphatidylinositol,phosphatidylglycercol, cationonic surfactants, such asdidodeceyldimethyl ammonium bromide (DDAB), monoglycerides, all of whichform lamellar phases in equilibrium with the interior phase and withexcess of solution; class 2) monoglycerides forming a lamellarcrystalline phase in equilibrium with the cubic and excess solutionphase; class 3) In addition to those given in procedure a) abovephosphatidylglycerols and phosphatidylethanolamine, both with chainlengths of 18 carbons or above and unsaturated, can be mentioned.Repeated freeze-thawing cycles can be used to control particle sizedistribution, and the dispersion obtained can be aftertreated asdescribed below.

All except procedure d) take place above the main transition temperatureof the lipid bilayer or lipid mono-layer constituting the interiorphase. Any variation of the procedures a)-d) which utilizes the natureof the exact potentials, i.e. uses another pathway so to achievesubstantially the same result as disclosed herein, fall within the scopeof the current invention.

3.1.1. Examples of procedures a) and b)

Materials: A GMO prepared by molecular distillation was purchased fromGrindsted Products A/S, glycerol monooleate (GMO) (85-06) (074832-FF8-009), (Braband, Denmark), and consisted of 98.8% monoglycerides, 1.0%glycerol, 1.0% diglycerides and 1.0% free fatty acids. The fatty acidcomposition was C16:0:0.5, C18:0:2.0, C18:1:92.3, C18:2:4.3,C18:3:trace, C20:4:0.5 wt. %, as stated by the supplier. Purifiedpoloxamer 407, also name, Pluronic F-127, was obtained from BASFCorporation (Wyan-dotte, USA). Soybean phosphatidylcholine (SPC) waspurchased from Lucas Meyr (Epikuron 200) with a fatty acid patternaccording to Rydhag (1979) of: C8:0.8, C12:2:12.2. C16:1:0.4, C18:2.7,C18:1:10.7, C18:2:67.2 and C18:3:6.0. Double distilled water was used inall experiments.

In the ternary phase diagram of the GMO/poloxamer 407/ water system thesolubility of the fragmentation agent, in this case poloxamer 407, inthe cubic phase originating from the binary GMO/water system is suchthat it introduces the formation of a new cubic phase which is inequilibrium with an L3 phase which exists between 78-90 wt. % of waterand an aqueous phase. These three phases form the boundary of theconstituents of the invention in this particular system, by the threephase region where the interior phase, the surface phase, and thesolvent-rich solution phase coexist. Thus only one particular cubicphase, a cubic phase Q²²⁹ with a lattice parameter of 15 nm and acomposition of 50/3.5/46.5 wt. % of GMO/poloxamer 407/ water,respectively, is fragmented by means of the introduction ofdisclinations caused by the local formation of the dispersable phase,the L3 phase, whose composition is 6.5/4/89.5 wt. % of GMO/poloxamer407/ water, respectively. Mixtures of the compounds whose composition issuch as it falls within the boundaries of the three phase region arereadily fragmented, even spontaneously with some fragmentation agentsacting by this mechanism. Such mixtures thus fall within the scope ofthe current invention.

The following procedures have routinely been used to produce theparticles of the invention: Typically an aqueous poloxamer 407 solutionwas added to a homogeneous cubic phase GMO/water (65/35 w/w %). Theamount of poloxamer 407 solution can be varied within the three phaseregion described, i.e., in the approximative range of 0.8-3.5 wt. % ofpoloxamer 407. When necessary, the mixture was stirred on a magneticstirrer until the cubic phase had fragmented into particles with thedesired properties, such as size and adhesiveness. Typically water wasadded to a powdered GMO in the ratio 65/35 w/w %. The mixture was thenequilibrated at room temperature for some hours until a clear isotropicphase was obtained, after which an aqueous poloxamer 407 solution wasadded according to concentrations given above.

Analogous behavior of the GMO/poloxamer 407/ water system is obtained ifthe poloxamer is changed to poloxamer 188 (Pluronic F68) instead of 407.In the GMO/poloxamer 188/ water system the three phase region in whichthe cubic phase constituting the interior of the current invention hasthe composition 53/4/43 wt. % of GMO/poloxamer 188/ water and the L3phase constituting the dispersable, or surface phase has the composition10/18/72 of GMO/poloxamer 188/ water. The three phase region in whichthe particles of the current invention can be produced is thus defined.

The fragmentation procedure itself, in these and the other systemspresented, requires very little input of energy, and fragments of theinterior phase are spontaneously formed by simply mixing the components.In all systems homogenizing devices, e.g., valve homogenizers can beused as described below.

The addition of any kind of compound which does not cause anyunfavorable phase change (unfavorable in the sense that none of theherein disclosed interior phases is formed) behaves analogously to thesystems described, and it is only the extension and location of thethree phase region which is changed, due to shifts in the phaseboundaries, of the phase regions constituting the interior phase as wellas the surface phase. For example, the system of GMO/soybean lecithin(SPC)poloxamer 407/ water is found to behave analogously to theGMO/poloxamer 407/ water and GMO/poloxamer 188/ water systems. The sameanalogy holds for a variety of other compounds as well, examplified bysomatostatin and insulin as described below.

In addition to the above systems the inventors found the formation ofthe current particles in: the GMO/DDAB/-water system, with less than 3wt. % of DDAB, in which particles with an interior phase of cubic phaseand an exterior of L3 phase are formed; systems ofdioleoylphos-phatidylethanolamine (DOPE) in combination with DDAB and orGMO, as well as diacylglycerides such as diolein.

3.1.2. Structure of the dispersion formed according to procedures a) andb): Evidence for an intraparticle cubic phase

The strategy in the structural evaluation of cubic phase dispersionsprepared according to procedure a) has been to use a combination of thefollowing: i) phase diagram studies; ii) detailed SAX diffractionstudies to complement the phase diagram studies and to verify directlythe existence of cubic phases; iii) visualization of the dispersedstructure by detailed light microscopy and electron microscopy (EM)studies; iv) 31P-NMR to warily the existence of isotropic signals in thedispersions, indicative of cubic or L3 phases; v) light scatteringstudies for the measurement of particle size distributions. All steps inthe evaluation procedure have been performed on homogeneous phases andon non-homogenized and homogenized dispersions, with the exception of EMwhich was not applied in the case of homogeneous phase studies. Theresults are summarized below.

3.1.2.1. The GMO/poloxamer 407 and poloxamer 188 and GMO/SPC/poloxamer407-water systems

From X-ray studies of the fine structure of the cubic phase swelled withpoloxamer the inventors have shown that it is built up of a bilayersimilar to the cubic phases in the GMO-water system but in which thePPO-units of the poloxamer 407 are located in the bilyaer close to theapolar/polar interface with the PEO-units in the aqueous labyrinths.From this it is concluded that the observed decrease in the bilayercurvature of the cubic phases appears to be an effect of the PPO-unitsrather than the PEO-units. It is further proposed that the formation ofthe colloidal fragmented dispersions of the cubic phase is an ultimateconsequence of the proposed fine structure and its relation to theformation of an L3 phase. Thus, the inventors have shown that theformation of the L3 phase which is governed by, among other things, amelting of the lattice structure and a simultaneous approximatelytwo-fold increase of the characteristic length scale along a cubic/L3tieline. This leads to a weakening of the interbilayer forces and asubsequent loss of long-range order. This is utilized in the currentinvention, in such a way that the formation of the L3 phase acts verysimilar to an explosive, in that it bursts the homgoneous cubic phase.The pathway for the explosive force is the formation of the L3 phase,which takes place along the most cost-effective energy-minimized path. Aparticle will, however, not separate from the homogeneous cubic phaseuntil the whole of its boundary is covered with L3 phase. This can verydramatically and readily be visualized in the polarizing microscope, inwhich the fragmentation procedure can be seen to take place under theformation of small streaks of birefringence along the cracks of thehomogeneous cubic phase in an otherwise totally isotropic picture.Sometimes, fragments are sticked to the homogeneous cubic phase whichindicates that the surface is not fully covered with L3 phase.

The phase diagram of the GMO/poloxamer 407/ water system is dominated byan extended cubic phase region ranging from 18 wt. % to about 67 wt. %water and with a maximum content of 20 wt. % poloxamer 407. At higherconcentrations of the poloxamer 407 the cubic phase "melts" into anisotropic L2 phase. The system exhibits four other one phase regions,one of which appears in the diluted region, in between a dilutedlamellar phase and the cubic phase, viz an L3 phase. Its structure hasbeen shown by the inventors to be the same as for the L3 phasesfrequently found in surfactant-water system (see 2.5.2).

Four cubic one-phase regions were verified experimentally by theexistence of the necessary two-phase samples with clearly separated,subsequently identified cubic phases, and the existence of the necessarymultiple-phase samples formed with the adjacent phase regions. The typeof the cubic phases may be assumed to be of type II based on theirlocation in the phase diagram. By means of SAXS the cubic phase whichparticipate in the three phase region where fragmentation takes placecan be determined. The same principle phase behavior is found in theGMO/poloxamer 188/ water and in the GMO/SPC/poloxamer 407/ watersystems, and the existence of a three phase region where the interiorphase (cubic) and the surface phase (L3) coexist with an excess ofwater. In addition we have studied the phase behavior of theGMO/poloxamer 188/ water at increased temperature to check for thecritical temperature when this three phase region disappears. No suchtemperature was found and the three phase region existed at least up to60° C. which was the highest temperature studied. A general decrease ofthe extension of the cubic phase was noticed to take place at high watercontent (low curvatures) which correlates nicely with the believe inincreased entropy in the hydrophobic part of the bilayer as thetemperature is increased. The most important feature for this text isthe existence of The three-phase region where the L3 phase and the cubicphase are coexisting with the diluted solution and in which region thecurrent particles may be prepared.

3.1.2.2. Phase behavior in polypeptide- and protein-water systems andtheir dispersions

The phase behavior of the GMO-somatostatin-water system is similar tothat of the MO/lysozyme/water system. Somatostatin, like mostpolypeptide hormones, has a very low solubility in water and tends toassociate into various types of molecular aggregates. The solubility ofmonomeric somatostatin (molecular weight 1637.9) in aqueous solution hasbeen estimated to be 0.3 mg/ml. Furthermore, it has a net charge of 4and a pI=10. The phase diagram of GMO-somatostatin at 20° C., clearlyshows an increase of the solubility of somatostatin when formulated inthe cubic phase. The cubic phase regions extend towards the wet regionmeaning that the curvature of the cubic phase(s) is decreased as theamount of somatostatin is increased. This reflects the amphiphilicnature of somatostatin analogous to the case of GMO/poloxamer 188 or amaximum of 10 wt. % of somatostatin have been studied. The limit ofswelling of the cubic phase is increased by nearly 10 wt. % as well.Several two-phase samples have been observed in the cubic phase regionand again there exists a complicated, yet not fully resolved, cubicmesomorphism. However, as with many other hydrophilic proteins andpolypeptides, we can conclude that the "average" curvature of thebilayer in the cubic phase is decreased as an effect of increasedsurface area of the apolar/polar interface caused by the presence ofsomatostatin. In line with this Q²²⁹ phase has been observed. We havealso investigated the phase behavior at 37° C., which shows the markeddecrease of the extension of the cubic phase at lower curvatures, i.e.higher amount of water. Again analogously to the results for theGMO/poloxamer 188/ water system. As seen a clear change of the phaseboundaries toward the "dry" region is obtained and this is compatiblewith an increased curvature as expexted.

3.1.2.3. Freeze-fracture electron microscopy study

We have also investigated the homogenized dispersions, prepared bymethods a) and b) given in section 3.1. and 3.1.1. and homogenized-asdescribed in section 3.3.1., with freeze-fracture electron microscopy(FFEM) in collaboration with W. Buchheim, Dept. of Chem. Phys., FederalDairy Research Centre, Kiel, Germany. Representative micrographs showthe characteristic pattern of a cubic phase enclosed in regular shapedparticles, often with square or rectangular cross-sections. The resultsare revealing, showing particles with a very characteristic appearance,and the structure of cubic phase is clearly seen. The periodicity of thecubic phase can be estimated to be about 150 Å, in good agreement withthe X-ray data. Furthermore, the average particle size is estimated tobe 300 nm, which is in the same range as obtained by the lightscattering experiment (see section 3.1.2.5.). From the largest particles(about 2 μm) a shift between adjacent fracture planes corresponding tohalf the periodicity could be seen, which indicates that the structureis body-centered, in agreement with the X-ray data.

The same results were obtained in the investigation of a fragmentedunhomogenized somatostatin cubic phase, used in section 4.1.4.1.

3.1.2.4. ³¹ P-NMR study

In order to investigate and further support the structural evaluation ofthe particles claimed an NMR investigation was performed. Dispersionswere studied by 31P-NMR by means of which it is possible todifferentiate between samples giving isotropic line shapes and thosegiving anisotropic shapes. The latter is characteristic of the lamellarand hexagonal phases, and the former of several isotropic phases, amongthem the cubic phase and isotropic solution phases. If the sample is amixture of two (or more) phases, the NMR spectrum is considered to be asuperposition of the individual signals (cf. Lindblom and Rilfors 1989).It is important to note that NMR is a nondestructive method in the sensethat it does not need separation of the individual phases.

Both homogenized and crude dispersions as obtained by the proceduresgiven in sections 3.1. and 3.1.1. and subsequently homogenized by theprocedure outlined in 3.3.1. were used. No significant difference couldbe observed between the two dispersions. Representative powder-typespectrum was recorded at resonence frequencies of 40.508 MHz on amodified Varian XL-100-15 spectrometer operating in a pulsed Fouriertransform mode. Only two rather closely located peaks, with an arearelation of 2:1, of narrow bandshape were observed. This indicates thepresence of fast isotropic motion i.e. isotropic phases. Even if it istempting to assume that these peaks correspond to the cubic and L3phases, it is important to stress that these peaks can arise from, e.g.,an L2 phase or vesicles, i.e., unilamellar liposomes. However, togetherwith the evidence obtained with the other methods, the presence of twosuch "isotropic" peaks supports the structure as deduced from the FFEMand X-ray studies.

3.1.2.5. Photon correlation spectroscopy (PCS) study

Homogenization of the coarse dispersion as described in section 3.3.1.results in a dispersion which shows enhanced colloidal stability, mainlybecause the reduction of particle size. Light scattering experiments(performed with a Malvern PCS100 spectrometer (Malvern Instruments Ltd.,UK) equipped with an argon-ion laser and K7032-OF correlator) indicatedthat the mean particle size was about 200-1000 nm, depending on thepressure, number of passages and temperature during the homogenizer usedin the experiments, as described in section 3.3.1. A representative sizedistribution of a homogenized dispersion shows the presence of very fewparticles bigger than 1 μm and about 90-95% of the particle size lies inthe interval of 150-500 nm with a mean of about 300 nm. This is inagreement with the results obtained by FFEM (see section 3.1.2.3.).

3.2. L3 phase-dispersions

These particles have been obtained through lyctropic phasetransformation of %he cubic phase dispersions described above. That is,the procedure is substantially the same, but the intention is that dueto the existence of an L3 phase in equilibrium with diluted solution(knowledge of which is obtained through prior phase diagram studies),the particle interior will be an L3 phase rather than a cubic phase.Systems where an L3 phase is known to appear in equilibrium with dilutedsolution are ternary systems containing amphiphilic proteins, such asthe casein/monoglyceride/water system, and the poloxamer 407/monoglyceride/water and poloxamer 188/ monoglyceride/water systems. Theaddition of lecithin, such as SPC, egg yolk lecithin, or lamellarforming cationic surfactants such as DDAB according to the aboveprocedures may favor the formation of these particles over the cubicphase particles, as may the increase in the concentration of the third,amphiphilic component. Thus, the production procedures are similar tothose described for fragmentation of cubic phases with The exception ofprocedure c) in section 3.1. The L3 phase particles are composed ofbicontinuous L3 phase interiors stabilized by the action of thepalisades created as described in a) and b) in section 3.1.

In summary some of the most important fragmentation agents can be foundwithin The following groups of compounds:

POLYMERS

Amphiphilic polymers:

amphiphilic block copolymers: amphiphilic di- and tri-block copolymers:pluronics (polyxamers) and tetronics: poloxamer 407 (Pluronic®F127),poloxamer 188 (Pluronic® F68); polyvinylpyrrollidine (PVP) amphiphilicproteins: glycoprotein, casein lipopolysaccharides: Lipid A andderivatives, analogs to Lipid A, and derivatives thereof.

diacyl lipids with polymeric polar heads.

Amphiphiles, including lipids, and lipid-like surfactant and derivativesthereof

Nonionic: polyethyleneoxide surfactants: n-alkylpolyglycol ethers (C_(i)EO_(j)); various derivatives of polyoxyethylene (POE): POE fatty amine,POE glycol monoethers, POE fatty ester, POE fatty alcohol; polysorbates;sorbin esters.

Anionic: alkylsulfates; soaps; sulfosuccinates.

Cationic: quaternary ammonium compounds (cationic soaps):cetyltrimethylammonium bromide (CTAB), didodecyldimethylammonium bromide(DDAB) etc.; N-[1-(2,3-dioleoyloxy)propyl]-N, N-trimethylammoniumchloride (DOTMA) and various analogs; cationic headgroup derivatives ofmonoacyl- or diacylglycerol.

Zwitterionic: phospholipids: phosphatidyl-choline (PC), -ethanolamine(PE), -serine (PS), -glycerol (PG): dioleoylPC (DOPC), dioleoylPE(DOPE), dioleoylPG (DOPG) (All C18:1 alkyl chains, but there are manyother examples and combinations); alkyl betaine derivative.

Lipid derivatives: polyethyleneglycol derivatized (phospho)lipids(PEG-PC and PEG-PE); ethoxylated cholesterol.

Glycolipids: mono-, di- and polyglycodiacylglycercls.

3.3. Aftertreatment and additional processing

Depending on the desired properties of the final formulation, selectedin view of the particular application, various aftertreatment processesmay be desired. Particles made according To procedure b) section 3.1.can be homogenized with preserved structure by means of suitableequipment, such as a valve homogenizer, so as to achieve a certainparticle size distribution. Other processes such as sterilization bymeans of an autoclave, sterile filtration, or radiation techniques, orcombinations thereof, may be applied with preservation of theintraparticle structure and of the physicochemical state of the activecompound, as now described.

3.3.1. Homogenization

Depending on the system in which particles, as described above, havebeen obtained and dependent on the desired properties, the dispersioncan be homogenized so as to achieve a satisfactory particle sizedistribution and surface properties. The decrease of particle sizeincreases the stability of the dispersion, with regard to settlingphenomena. However, it is important not to treat the destabilizationprocesses with normal procedures for emulsion system, since the currentsystem is not acting as such, nor can it be defined as such. Theassociated destabilization is treated in more detail in section 3.4.3.

After fragmentation of the cubic phase the dispersion may have differentsurface characteristics depending on the fragmentation agents used. Thisin turn affects particle size and properties such as adhesiveness, andit can be advantageous to homogenize the dispersion for reduction inparticle size and a narrow particle size distribution. In particular,homogenization is important in the use of the current particles as drugdelivery system.

In practice several different homogenizers may be used; however, theintroduction of new equipment requires a thorough structural evaluationso as to insure that the particles still have the intraparticularproperties of the cubic phase as described above. In this work we haveused two principle devices to homogenize the dispersions: an ultrasonicdevice and a valve homogenizer. The homogenizer used is described indetail by Thornberg and Lundh (1978). Briefly it is a pneumaticcontinuous valve homogenizer for laboratory use equipped with a heatexchanger and a valve. Its capacity can be varied as well as itspressure, which can be monitored. Usually, batches of 10-50 ml wascontinuously homogenized at 25° C., with pressures applied in the rangeof 80-180 kBar. The dispersion was carefully investigated during andafter the homogenization.

Preparation of homogenized dispersions used for FFEM, 31P-NMR, and PCSstudies: A batch of the GMO/water cubic phase was prepared, sealed andstored under nitrogen atmosphere at room temperature. The water contentwas checked by frequent ocular examination. Normally dispersions wereprepared in samples of 30 ml, with a total lipid composition of 10 w/w%, and final concentrations of the GMO/SPC/poloxamer 407 system of6.5/3.5/1.0 w/w %, st. These dispersions were prepared with theGMO/water cubic phase as starting material for the dispersions. Asuitable amount of the GMO/water (65/35 w/w %) cubic phase was weighedinto a ordinary beaker and mixed with appropriate amounts of a premixeddispersion of poloxamer 407 and SPC in accordance with the finalconcentration. The mixture was stirred for some hours until the coarsedispersion was suitable to homogenize. The homogenization was carriedout at 25° C. in continuous laboratory valve homogenizer, described indetail by Thornberg and Lundh (1978). Immediately after homogenizing thedispersions were collected in 12 ml ampoules which were filled withnitrogen and flame sealed. All dispersions were stored at roomtemperature.

The increase in stability is indicated by the fact thatultracentrifugation at 250,000 xg (20° C., 24 hr) was not enough toseparate the pases satisfactorily as compared to 40,000 xg (20° C., 24hr), at which a coarse dispersion of a corresponding sample wasseparated. By examination of samples after each passage through, thevalve, both in the microscope and by X-ray experiments, performed afterseparation of the dispersion by means of an ultracentrifuge, it wasdeduced that no apparent changes of the structure, other than sizereduction, had occured.

All the steps of the dispersion procedure were carefully followed byocular inspection and by light microscopy observations, looking forsigns of birefringence and estimations of the particle size. Thestability of the dispersions was judged by ocular inspection.

3.3.2. Sterilization

Due to the stablization by the fragmentation agent, the sterilizationmethod has to be carefully established for each system underinvestigation. Particles of the cubic phase dispersion type stabilizedwith poloxamers according to above might, however, be sterilized byseveral methods without affecting the final structure or the physicalproperties of the particles. This is due to the very high inversiontemperature and the very high cloud point temperature (or lack thereof)for these amphiphilic block copolymers. Similar properties can be foundto be valid for other block copolymers listed as fragmentation agentsabove. Thus, these formulations can be sterilized by autoclavingtechniques with the consideration of peroxidation carefully evaluatedand taken into account.

For the majority of dispersed systems sterile filtration is the onlycurrently acceptable method available. Techniques such as thosesuggested and currenctly used in the field of biotechnology, inparticular liposome and emulsion technologies can be used in connectionwith the current invention.

3.3.3. Further stabilization

Destabilization procedures due to the colloidal nature of the currentparticles may in principle be avoided by the same methods used in othercolloidal systems, such as emulsion and dispersion technology. Inparticular, addition of polymers such as alginates, amylopectin anddextran, may enhance stability, as well as the use of stericstabililizing fragmentation agents.

3.3.4. Freeze drying

Protein cubic phases can be freeze dried with retained protein structure(enzyme activity) after reconstitution (Ericsson 1986). Thus, disclosedstructures as reported here can also be freeze-dried and reconstituted.Such preliminary results have been performed and X-ray diffraction datashow no significant difference between the original dispersion and thereconstituted dispersion. In particular, such procedures as freezedrying can be performed with cubic phases containing substantillayamounts of sugars, such as (Soderberg 1990) which is thought to protectthe tertiary structure of proteins and possible other compounds asnucleotides.

3.4. Variations of methods of preparation

Formulations such as those exemplified above in general may have to bemodified and processed in such ways that formulations fulfil thecriteria set by the government. In particular, additives, such asglycerol, sucrose, phosphate buffers and saline in relevantconcentrations, to the aqueous compartment or formulations thereof canbe added wihtout changing the principle structure of the particles.

A particular feature of the dispersion made by method a) section 3.1. isthat it is stable with the same principle intraparticle structure withina pH range of approximately 2-9. Similar pH stability ranges can beexpected for other non ionic systems. The addition of charged lipidspecies, or active ionic compounds, in the formulation can thus be usedas a pH sensitive releasing/triggered system.

3.4.1. Solvent-based methods

The processes described in sections 3.1. and 3.2. can be variedaccording to the following: by solvent solubilization of the componentscorresponding to the constitution of the cubic phase as described in 3.1and 3.2 or such solubilizate plus bioactive agents as described insetion 4, or such solubilizate with or without bioactive component plusfragmentation agent as described in sections 3.1 and 3.2. Solvents thatcan be used are, DMSO, carbontetrachloride, ethanol, methanol, hexane,or mixtures thereof. After subsequent evaporation of the solvent a cubicphase is formed. Evaporation can be achieved by conventional methodssuch as a rotavapor. Similar methods currently in use in the field ofliposome preparation technology can in principle be applied to thecurrent invention. The cubic phases are subsequently treated asdescribed in section 3.1. for the formation of the dispersions throughfragmentation of the phase constituting the interior phase, or section3.2. for the formation of the L3 phase dispersion.

3.4.2. Dispersions with polymerizable lipids or lipid-like surfactant

Another possibility that lies within the scope of this invention isafforded by cubic phase-forming surfactants, lipids, and amphiphilicmonomers that can be polymerized. In particular, those techniques whichare disclosed in the following Anderson documents, concerning thepolymerization of cubic and other reversed liquid crystalline phases,are in principle applicable to the present invention for theestablishment of a polymrized interior phase, surface phase, or both:Anderson, D.M. (1990) Coll. de Phys. 51(23) C7-1. Anderson, D.M. andStrom, P. (1991) Physica A 176, p. 151; Anderson, D. and Strom, P. (1989in Polymer association structures: microemulsions and liquid crystals(El-Nokaly, M.A. ed.) pp. 204, American Chemical Society, Wash. Strom,P. and Anderson, D.M. (1992) Langmuir 8, 691.

Research has recently arisen in the literature as to polymerizablelipids that polymerize through peptide bonds. Cubic phase particles madeand polymerized with such lipids could be processed in particulardepolymerized --through polypeptide degradation and biosynthesispath-ways in the body. In this way, sites of excessive or abnormalpolypeptide metabolism could be targeted.

The use of polymerizable (or polymerizable/depolymerizable) compounds asdispersing agents opens up the possibility to tailor the characteristicsof the palisade layer, substantially independently of those of theinterior. In particular, with minimal effect on the cost effective,protein stability, and microstructure of the interior of the particles,the hydrophilic palisade could be polymerized with a resulting strongeffect on the stability and molecular recognition properties of theparticles. The release rate, and even the functional form of theprofile, could be tailored in this same way, especially through the useof mixtures of polymerizable and non-polymerizing agents or agents withvariable numbers of polymerizable groups, establishing distinguishablelabyrinths through polymerization of chiral and monolayer cubic phases.In addition to triply-periodic order, an additional degree of spatialorder is afforded if the two solvent networks created by the surfactantfilm can be independently treated, in a systematic way. We havementioned that more sophisticated applications of these materials couldbe made possible if this distinction were possible; that this is inprinciple possible, and of potential importance, is clear from the useof this property in the prolamellar body of etiolated leaves, forexample (Gunning 1967, Tien 1982). In this subsection we discussedseveral ways in which this might be accomplished, based on previous workof this group.

As discussed in section 2.4.2, the Q²¹² cubic phase structure, which hasbeen found in the monoolein/water/cytochrome-c system, is believed to bethe same as the structure of Q²³⁰ except that the protein is segregatedinto one of these networks by virtue of its stereochemistry; thus thecubic phase has one aqueous network and one network filled in withinverted micelles containing proteins.

4. Applications

Especially interesting uses of the particles claimed, or as prepared bythe method claimed, are those uses which are defined in the accompanyingclaims. These and other uses will, however, be described more in detailbelow.

In the area of drug delivery, the invention is particularly well suited,though not limited, to the formulation and delivery of hydrophobic andamphiphilic compounds that have limited aqueous solubility, or aresubject to undesireable degradation or non-optimal presentation to thetarget, especially, coformulations of nucleic acids and/or proteins withcompounds related to, or needed for, the uptake, introduction ortranscription, or for its enhancement, of nucleic acids. In addition,the invention is in principle well suited for intracellular targeting.The invention is well suited as an adjuvant for vaccines, such aslipopolysaccharides, particularly for peptide- or carbohydrate-basedantigenic compounds and in the colormulation of immunomodulators. Theinvention is well suited for prolonged circulation of peptidic drugs,and more particularly it increases the therapeutic index thus decreasingsystemic toxicity, which is common among compounds under investigationin the treatment of cancer and in the therapy of immune disorders, suchas HIV related diseases.

Thus a drug delivery system should protect the polypeptide fromdegradation as well as increase the half-life so to achieve longercontact times for site-specific and chronospecific delivery, bothextracellular and intracellular.

4.1 Drug delivery

A major obstacle for the utilization and delivery of polypeptides andproteinaceous active agents is their formulation. The simplest form ofadministration of these compounds is by direct injection in hypotonicmedium into the bloodstream. However, several properties of polypeptidesand proteins that may impede their delivery must be taken inconsideration, such as their: i) physicochemical state; ii) chemical,enzymatic and physical instability; iii) short biological half-life ofcirculating compound; iv) potential of provoking immunological response;v) inability to be transported from the vascular compartment toextravascular sites with efficiency; and vi) the chronicity of theirbiological task.

The current invention represents a novel approach that circumvents theselimitations, and furthermore provides unique means by which to achievechrono- and site-specific delivery, including delivery specifically tointracellular sites--that is, directly to the cell organellesresponsible for the activation and control of biosynthetic pathwaysgoverning cell metabolism and dis-semination of genetically-derivedinformation. The current invention is not restricted to any particularroute of administration, and administration can be made by intravenous,intramuscular, intranasal, ocular, sublingual, subcutaneous, oral,rectal, vaginal, or dermal routes, or regionally such as throughintraperitoneal, intraarterial, intrathecal and intravesical routes.

4.1.1. Toxicological considerations

A problem with the use of homogeneous reversed cubic phases as drugdelivery system is its well documented fusogenic property and as aneffect it is hemolytic. The documentation is particularly well regardingmonoolein (see e.g. Cramp and Lucy, Hope and Cullis 1981 and referencescited therein). On the other hand there are reports regarding antitumoractivity of certain monoglycerides (Karo et al. 1969) as well asantimicrobial effects (see e.g. Yamaguchi 1977). The current particles,prepared by the methods given in section 3.1. and 3.1.1. do not show anyfusogenic activity even when the phase constituting the interior isoriginating from the GMO/water cubic phase, as was indicated by theabsence of hemolytic products in mixtures of a cubic phase dispersionand human whole blood. The apparent absence of toxic effect in theanimal test discussed herein also supports this conclusion, althoughclearly more testing is necessary. This apparent lack of toxicity isalmost certainly due to the increased hydrophilicity of this cubic phasedispersion as compared to the homogeneous cubic phase, and throughsteric stabilization, both provided by the hydrophilic palisade createdby the surface phase.

4.1.2. Site-specific drug delivery

It has become urgent to develop more site-selective and specificallytargeted drugs. In particular, the very potent and often systemicunwanted actions of peptidic drugs require efficient targeting so as toavoid the otherwise extremely high doses which can cause, e.g.,immunogenic responses. Furthermore, many of the endogenous peptidicsubstances considered as drugs act within 1-10 nm of their site ofproduction. Together with their variable efficiency with time of actionand the fact that polypeptides are rapidly metabolized, this clearlyrequires specific targeting to obtain and maintain relevant dose levels.Whether this will be achieved with the use of carriers, vehicles, drugdelivery systems, or the de novo synthesis of macromolecular therapeuticsystems is still an open question. Site-direction in drug delivery canbe obtained by using the endogeneous routes offered. These have onlybeen partially mapped today and perhaps the most explored pathway toobtain site selectivity has been to use monoclonal antibodies. Thesurface phase of the particles of the current invention can in principlebe tailored so as to be used in all kinds of different interactions withe.g. tissues, so as to increase the efficiency of the drug via theachievement of site-specific delivery and thus increase its therapeuticindex.

4.1.3. Organ selectivity

The use of sepcific fragmentation agents in the method of the currentinvention to achieve specific: interactions with a set or subset ofcells within one organ can e.g. be achieved through the use of differentamphiphilic block copolymers, such as the poloxamers 407 and 188. It hasalso been shown that the biodistribution can be altered by the differentsurface properties obtained by the use of these polymers. Thus, assuggested by these works, the cubic or L3 dispersions can be directedtowards, e.g., the bone marrow by applying different fragmentationagents as described in section 3. The use of carbohydrates orsynthetically modified block copolymers as fragmentation agents can beused in the present invention to further increase the specificity. Themodification of the hydrophilic polyethyleneoxide units by conjugationwith specific sugar moleties can be used as one approach.

The use of lectins (Sharon and Lis 1989) offers an attractive pathway tospecific interactions with targets. It has been demonstrated thatmembrane bound lectins mediate the binding of both cellular andintracellular glycoproteins to membranes and in this way control thetrafficking of glycoproteins. It is clear that this highly conserved andspecific interaction can be used the: other way around, i.e. that theglycoprotein (or only mimic thereof) is carried by the vehicle and uponrecognition by the lectin interact with the target. Again the currentinvention offers important novel possibilites to achieve a uniform andefficient presentation and interaction with the target.

4.1.4. Delivery of polypeptides and proteins

The success of recombinant products for use as pharmaceutics will, atleast in part, be dependent on progress in the formulation of theseproducts. However, due to the great variation of these products withregard to their physicochemical properties and biological action, atheir physicochemical properties and biological action, a singledelivery system is very unlikely to satisfy all the desired properties.E.g. in the formulation of polypeptides it is often thought that asustained release should increase the bioavailability; however, thereare many examples where a sustained release could cause toxic orimmunogenic reactions due to, e.g., cascade effects. Clearly, the doseresponse and the dose determination rely on several complicated issues,from the development of international standards to the more basicunderstanding of the biochemical action of these products, and, stillmore intriguing, the patient dose dependence. Depending on the time andduration of their interaction with the target, the range of effects alsomight be selective. Thus, chronospecificity is also to be considered,particularly in the delivery of peptide hormons or neuropeptides.

For some polypeptides a duration of their delivery and a prolongation oftheir biological half-life may be of relevance and increase thebioavailability and/or efficiency. Currently most formulations ofpolypeptides have been concerned with the rather trivial question ofincreasing the biological half-life upon administration. Preparationssuch as liposome-associated polypeptides have been used to sustain thedelivery of many polypeptides through various routes, and to some extentit has been shown that the delivery of intact and bioactive polypeptidescan be prolonged for days and possibly longer. However, very few ofthese studies have been at all concerned with the therapeutic efficiencyof the delivery. This aspect will, however, determine whether productsas liposomes will be of medical or commercial importance in a specificapplication. The numerous obstacles to the efficient delivery ofpolypeptides and proteins have been considered by many authors (Sternson1987, Lee 1988, Eppstein and Longenecker 1988, Banga and Chien 1988).

Peptidic drugs comprise a broad class of pharmaceutics and the in vivoactions of these compounds, whether administered or endogenous, includea wide range of effects; due to their interactions, these areintrinsically coupled, and therefore some of the peptidic drugs aretreated separately elsewhere in this text. Thus to understand andcontrol systemic effects, site-specific delivery must be consideredalong with the above-mentioned considerations.

Coformulations of absorption enhancers such as bile acids, for examplesodium glycocholate and deoxycholate, as nonionic polyoxyethylene ethersand derivatives of fusidic acid such as sodium taurodihydrofusidate, ora combination of these, are easily achieved with the current invention.

For some peptidic compounds precautions have to be taken to avoidprecipitation, fibrillation, and/or aggregation of the compound. In thecurrent invention such changes are most conveniently avoided by addingthe peptidic compound as a solution to a preequilibriated cubic phasewith the smallest possible amount of water within the cubic phase regionso as to swell the cubic phase to the cubic phase considered for thefinal equilibration and subsequent fragmentation, or by adding thepeptidic solution to a preequilibriated mixture of lamellar and cubicphase, i.e. to a two-phase region consistent with the coexistence atequilibrium conditions of lamellar phase and cubic phase in accordancewith phase behavior, followed by equilibration and subsequentfragmentation. The latter is exemplified in the GMO (or MO or mixturesof monoglycerides or mixtures of certain monoglycerides andphospholipids or lecithin)/water system.

Examples of peptidic compounds which can be formulated with the currentinvention are: bovine serum albumin (BSA), insulin, epidermal growthfactor (EGF), gonado-tropin-releasing hormone, interferons (type I andII: I:alfa (15-20 different species) and beta; II: gamma), luteinizinghormone, vasopressin and derivatives, somatostatin and analogs. Otherpeptide-based pharmaceuticals that are active in the cadiovascular, CNS,and gastrointestinal regions, as well as those modulating the immunesystem or the metabolism can be formulated either alone or in mixturesby the current invention.

4.1.4.1. Example of intravenous somatostatin formulation in the rabbit

Somatostatin is an endogenous small polypeptide with a wide range ofbiological effects which have been subject to intensive researchactivity since it was discovered in 1968. The very broad spectrum ofactions of somatostatin have led to an immerse search for therapeuticapplications. However, because of its low biological half-life, greatefforts have been put into the development of more stable and specificanalogues more suitable for clinical applications.

In order to investigate the properties of cubosomes as a drug deliverysystem, a somatostatin loaded cubic phase dispersion preparation wasstudied in the rabbit using intravenous bolus injection. Afterinjection, blood was sampled at regular intervals, and concentrations ofsomatostatin were determined as the specific immunoreactivity in plasma.It is important to note that these measurements do not reveal the amountreleased, and the amount of somatostatin measured can very well belocated in the cubosomes. This is a common analytical problem shared byother drug vehicles. The result showed a significantly increased andmaintained somatostatin concentration in the plasma within the time ofthe experiment which was six hours. Also shown is the plasmaconcentration after bolus injection of the peptide. From these resultswe can conlude that the cubosomes exhibit a prolonged circulation time.This can tentatively be explained by the palisade of PEO-units whichincreases the hydrophilicity. This effect has been observed for othercolloidal drug carriers covered with a surface layer of poloxamer. Infact several studies have used poloxamers to prolong the circulatingtime of colloidal particles via an adsorption of the block copolymer tothe surface of the particles, resulting in an increased hydrophilicityof the particle surface (cf. Jamshaid et al. 1988 and references).

4.1.4.2. Example of intranasal insulin formulation in the rat

Insulin (Actrapid, Novo, Denmark) has been formulated by the currentinvention and delivered intranasally in a rat. Insulin solution with thedesired concentration was added to samples of GMO/water, correspondingto the two-phase region where the lamellar and cubic phase (Q²³⁰)coexist, following equilibration to insure the formation of the desiredcubic phase used for subsequent fragmentation. SAXS methods were used toinsure that the formulation prior fragmentation correspond to a cubicphase. Fragmentation was performed as described in section 3.1 method b)as described in detail in section 3.1.1. All steps in the formulationprocedure were performed under antiseptic conditions. In the currentexample with regard to route of administration and the delicateproperties of insulin the fragmented colloidal dispersion was chosen notto be further processed. There were no signs of instability of thedispersion during the time of the experiments (approximately 2 months).

Animal experiments were performed in collaboration with Dr. P. Ederman,Dept. of Pharmacy, Uppsala University. The formulation (10 IU/ml) wasadministered intranasally in rats (Wistar) and the change in the bloodglucose was used as a measure of the insulin delivery through the nasalepithelia. A significant change was observed and no obvious signs ofside effects were observed.

4.1.5. Adjuvant formulations and the use of the current particles as avehicle for immunomodultative compounds

The development of specific recombinant or synthetic antigeneticsubstances, such as subunit antigen and polypeptides, have lead to anurgent need for vaccine adjuvants since these structures are generallyof low antigenicity. Therefore their promise as vaccines will rely ontheir formulation with adjuvants that increase cell-mediated and humoralresponses (Alison and Byars 1990).

The two most often considered lipid-based colloidal vaccine adjuvantsare liposomes (Allison and Gregozriadis 1974, Gregoriadis 1990) andFreunds adjuvants (Edelman 1980). Lately, however, the use of liposomesas adjuvant systems has been reconsidered and as pointed out by Weiner(Weiner 1989) liposomes do not insure an increased immune reactivity.The properties and features of some new adjuvants for vaccines have beenreviewed (Eppstein et al. 1990).

It has become clear that the presentation of the antigenic structure isof great importance for the immune reactivity, response and thesubsequent development of immunity and that membrane perturbations areof profound importance in immunogenic responses. Even though notgenerally accepted it is believed that the events upon membraneperturbations are intimately connected to the required intracellularenzyme activity and that the rationale for this is the cooperativebiogenesis of intracellular membranes and especially the expression andsurface properties of the plasma membrane. It is therefore veryinteresting to note that the biological active part of differentlipopolysaccharides (LPS), Lipid A, forms a cubic phase in excess ofphysiological solution at physiological conditions (Brandenburg et al.1990) and thus behaves very similar to the monoolein-water system. Itcan also be fragmented by the methods given in section 3 and used eitheralone or with solubilized vaccines and/or other immunomodulatingcompounds. Lidid A and synthetic analogous have frequently been used inadjuvant formulations for immunostimulation. It is therefore to beconsidered that coformulations of monoolein-lipid A forming a cubicphase in excess of solution would elicit strong immunogenic response.Other coformulations either with GMO, Lipid A or a combination of these,are with saponins (Barla et al 1979), bile acids (Lindstrom et al. 1981,Svard et al. 1988), phospholipids (Gutman et al. 1984) anddiacylglycerols.

In particular, the particles of the current invention are suitable as anadjuvant for polysaccharide antigens. It is well established thatadjuvants, such as LPS's and muramyl dipeptide (MDP), induce theproduction--by accessory cells--of mediators, such as interleukin-1(IL-1) and other lymphocyte growth factors, stimulating theproliferation of helper T-lymphocytes. The importance of the targetingof antigens to highly efficient presenting cells, such asinterdigitating and follicular dendritic cells, has been emphasized(Allison and Byars 1986). Thus, procedures that facilitate the migrationof antigens to the paracortical areas of lymph nodes, in the immediatevicinity of interdigitating cells, should favor cell-mediated immunity.

The particles of the current invention could thus be transferred toprecursors of interdigitating cells at the site of injection, inafferent lymph or in lymph node sinuses.

Similarly, any procedure, such as selective receptor targeting directedtowards C3b receptors on B-lymphocytes, that facilitates localization ofantigens on follicular dendritic cells should increase B-lymphocyteresponses.

Therapeutics which have been investigated as differentiating orinhibiting compounds are very attractive candidates to formulate by thecurrent invention. In particular, peptidic drugs such as oligopeptidesmay act in the inhibition of EGF receptor. Similar approaches arecurrently investigated in the treatment of the acquired immunodeficiencysyndrome (AIDS), a subject dealt with in section 4.1.7.1. Anotherpotential application is the coformulation of the antigen and/orimmunomodulator with cytostatic drugs such as methotrexate, so as topotentiate the immunogenic response and/or decrease or diminishpotential immune responses to antigens in antibody-targeted applicationsof the current invention.

4.1.6. Cancer therapy

The current invention should be applicable to the field of cancertherapy. As to further details in this context reference is made to theuse of liposomes for such purposes (see e.g. Weinstein 1987).

Preferential delivery to tumors is a challenge that, if solved, wouldrepresent an enormous advanve in cancer therapy. Reduced systemicconcentrations of the drug being delivered is a step is this direction,and by applying the methods described in section 4.1.2. and 4.1.3, siteselectivity could in principle be realized. Lipid biogenesis and therole of lipids as second messengers in the development of cancer hasdrawn increased research interest and activity. Coformulations ofanticancer drugs and such second messengers can easily be accomplishedby the current invention.

The rationale for use of lipid-based vehicles in general, and thecurrent invention in particular, as carriers in chemotherapy is based onthe following basic concept: prolonged circulation as compared to thefree drug; protection and stabilization of the drug; circumvention ofcertain cell membrane barriers; amplification of the drug effect due totargeted drug delivery. Agents of particular interest in this connectionare: doxorubicin; taxol; alopacia; cisplarin derivatives; andvincristine.

4.1.7. Antimicrobial therapy

The potency of drugs used against microbial infections, i.e.bacterial/rickettsial, parasitic and vital infections, can be correlatedwith their lipophilicity. The more lipophilic the more potent. Thus, thecurrent system can solubilize large amounts of potent antimicrobialagents and at the same time protect or minimize the host from systemictoxic effects.

Many of the new antimicrobial drugs under consideration are ofproteinaceous type. Some of these polypeptidic drugs considered fallalso in the category of immunomodulators, such as some interferons.Further examples in this category are lymphokines for the treatment ofvisceral leishmaniasis, and cytokines; ampicillin for the treatment ofintracellular liver and spleen infections caused by Listeriamonocytogenesis; amphotericin B in the treatment of mycotic infections;ribavirin for the treatment of fever virus infecton; streptomycin forthe treatment of tubercolosis and splenic infections; sisomycin for thetreatment of lung infections; gentamicin for the treatment ofintraperitoneal infection; and penicillin G for the treatment ofintraperitoneal extracellular infection caused by Staphylococcus aureus.Applications where sustained release are of particular interest are inthe treatment of genital papilloma virus infections. Coformulations oflipophilic, amphiphilic and hydrophilic antimicrobial drugs can beachieved with the current invention. Such formulations find theirapplication in the treatment of patients of systemic microbialinfections, which often is the case for patients having AIDS.

4.1.7.1. Therapy of human immunodeficiency virus-related disease

The potential for the use of the present particles in a delivery systemfor therapeutic intervention in therapy of human immunodeficiency virus(HIV)--related disease, especially acquired immunodeficiency disease(AIDS), follows from their potential in polypeptide and proteindelivery, in the delivery of immunomodulative compounds, and inintracellular targeting and delivery of such compounds, as discussedherein. Of particular importance in this respect is their potential usein the modulation of lipid biogenesis and the subsequent membraneformation, as target for the treatment of, e.g., cancer and otherhyperactive cells such as virus-infected cells.

Selective drug delivery is importance to cells which are infected or tocells which are known to be targets for HIV infection, such as CD4+Tcells, certain types of B cells, monocytes/macrophages, dendritic cells,Langerhans cells, and some brain glial cells, as well as HIV infectsCD8+T cells, muscle cells, fibroblastoid cells, and some neuronal cellsin vitro. Thus, all these, and other, cell types are potential targetsfor the current particles. Indeed, the same rationale as outlined forthe targeting of immunomodulators and vaccine formulations are valid inmost, in particular, the high surface area and the ability ofcoformulation.

The formulation of drugs or the use of fragmentation agents or partthereof which binds to target cells or preferably HIV, thus enablesinhibition of gp 120 induced cytocidal effect. Such compounds are CD4analogs and antibodies to HIV. The use of the current particles benefitssuch interactions needed and their prolonged circulation increases theseinteractions.

The (site specific) delivery of reverse transcriptase inhibitors, suchas 3'azido-2'3'-dideoxythymidine (AZT) and other dideoxynucleosideanalogs such as ddI, d4T and AZddu using the current nomenclature,dipyridodiazepinone derivatives andtetrahydro-imidazo[4,5,1-j,k][1,4]-benzo-diazepin-2(1H)-one (TIBO) and-thione(tibo) and derivatives.

The (site specific) delivery of specific inhibitors of HIV ribonucleaseH (RNase H) activity, inhibitors of transcription and translation of thevirus encoded replication.

Formulations with compounds that block the processing of the virusenvelop glycoprotein gp 120 and/or gp 41 or their precursor, such asN-butyl-nojirymycin.

Coformulations of certain immunomodulators or inducers thereof such asinterferons, in particular IFN-gamma, with other compounds used in thetreatment of HIV infections.

4.1.8. Gene therapy

The rationale of gene therapy has been discussed by many authors (cf.Wilson 1986) and the field appears to have a high potential. It is verylikely that the efficiency and stability of the introduced gene willdepend on its specific delivery to the target cell and on intra-cellularinteractions. For example, in the treatment of bone marrow cells, anextraordinarily efficient and selective delivery is required since stemcells of the bone marrow only comprise about 0.001-0.01% of the totalcells. The task to be solved by the delivery system for in vivotargeting is therefore considerable. The current invention offers notonly a very high solubility of DNA fragments and/or plasmids but alsoprotects DNA from undesireable interactions with body fluids.

4.2. Biotechnological and biomedical applications,

The considerable direct, and indirect, evidence for the high enzymaticactivity in the cubic phase particles of this invention, together withtheir versatility in the immobilization of enzymes at high loadings makethe current invention advantageous as compared to current availableimmobilization units in biotechnology. This has been discussed in thecase of homogeneous cubic phases by Anderson (1987), and the currentinvention represents an improvement of that invention because of themuch higher surface/volume ratio in submicron particles of the cubicphase, thus facilitating access into and out of active sites. The use oflipid-based vehicles in imaging, in particular liposomes ascontrast-enhancing agents or in diagnostic nuclear medicine have beenreviewed (Weinstein 1987, Caride 1985). Applications of the currentparticles in diagnostic imaging are as carriers of radiological contrastagents.

Delivery of oxygen can be achieved by the preparation of an oxygencarrier, such as the heme group in hemoglobulin or similar protein, in acubic phase. Cubic phases in the system hemoglobulin-GMO-water have beeninvestigated and are formed with high amounts of protein (>5 wt. %).Such a system can be used as blood substitute and in connection withradiation therapy of cancer. The use of polymerizable lipids, in suchsystems as described above could be used to enhance stability andshelf-life.

4.2.1. Transfection technology

The present invention should also be useful in the fields oftransfection technology, i.e. the introduction of foreign nucleic acidsinto cell types/lines. Particularly, in view of the currently usedliposomal system for the use in this field, such as DOPE which is aprominent non-lamellar forming lipids in aqueous systems, and the factfound by the inventors that the current particles can be formed in theGMO/DDAB/water system (see section 3.1.1.).

4.2.2. Cell culture

The invention should also be useful in the field of cell culture,especially use of the particles as carriers of nutrients, such as aminoacids, cholesterol, unsaturated fatty acids, but also as deliverysystems for more sepcific proteins as immunomodulators, growth factorsetc., or for the use as diagnostics, biosensor, in immuno-assays in cellculture. Also in the field of cell hybridoma technology applications arepossible.

4.3. Other applications

The present invention should also be applicable to other areas, inparticular applications in the area of biosensors and as catalyticparticles or carriers of catalysts. Other biomedical andbiotechnological areas include enzyme therapy (as with superoxidedismutase), dispersions with magnetic properties, immobilizaticn of theparticles in gel matrices. These particles can also provide sites formineralization and crysatallization. Mineralization of a substantialportion of the porespace or porewall surface could create microporousparticles with high chemical and thermal stability, and the use ofconducting or piezoelectric minerals or crystals could be important. Thepresence of polar groups at the porewall surface makes these particlesparticularly well suited for mineralization.

The present invention will also find cosmetic applications. Indeedexamples of the molecular constituents of the particles such asmonoglycerides and poloxamers are frequently encountered in cosmeticpreparations.

A curious phenomena found by the inventors is the capability of certaincubic phases, such as those formed in the GMO/poloxamer/water systemsdescribed above, to host fungi and also supply needs for their growth.Perhaps, most surprisingly the cubic phase maintains its characteristicsup to several months before phase transformation takes place. Thus thefungi grow inside the cubic phase without perturbing its structure.Therefore, the current particles could be utilized as culturemedia/machinery for the controlled culture of single, or multiplemicroorganisms.

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We claim:
 1. Particles comprising(A) an interior phase of (1) anon-lamellar lyotropic liquid crystalline phase selected from the groupconsisting of a reversed cubic liquid crystalline phase and a reversedhexagonal liquid crystalline phase, or (2) a homogeneous L3 phase, orany combination thereof, and (B) a surface phase (1) selected from thegroup consisting of a lamellar crystalline phase and a lamellar liquidcrystalline phase, or (2) an L3 phase, or any combination thereofwherein said surface phase and said interior phase are distinct. 2.Particles according to claim 1, wherein the interior phase is a reversedcubic liquid crystalline phase, a reversed hexagonal liquid crystallinephase or a mixture of reversed cubic and hexagonal liquid crystallinephase and a reversed cubic-hexagonal liquid crystalline phase and thesurface phase is an L3 phase.
 3. Particles according to claim 2, whereinthe interior phase is a reversed cubic liquid crystalline phase and thesurface phase is an L3 phase.
 4. Particles according to claim 1, whereinthe interior phase is a reversed cubic liquid crystalline phase, areversed hexagonal liquid crystalline phase or a mixture of reversedcubic and hexagonal liquid crystalline phase and the surface phase isselected from the group consisting of a lamellar crystalline phase and alamellar liquid crystalline phase.
 5. Particles according to claim 1,which contain added bio-active agents, or precursors thereof. 6.Particles according to claim 5, wherein said bio-active agent, orprecursor thereof, is selected from the group consisting of peptides andproteins, or protein-aceous compounds.
 7. A method of preparingparticles from a homogeneous liquid crystalline phase or L3 phase, whichcomprises forming a homogeneous, non-lamellar lyotropic liquidcrystalline phase selected from the group consisting of a reversed cubicliquid crystalline phase and a reversed hexagonal liquid crystallinephase, or a homogeneous L3 phase, or any combination thereof, creating alocal dispersible phase, within said homogeneous phase, of a phaseselected from the group consisting of a lamellar crystalline phase and alamellar liquid crystalline phase, or an L3 phase, or any combinationthereof, in the presence of a solvent phase, said solvent being of anature with which said homogeneous phase can coexist and wherein saiddispersible phase can be dispersed, and fragmentating said homogeneousphase so as to form particles, the interior phase of which comprisessaid homogeneous phase and the surface phase of which comprises saiddispersible phase.
 8. The method according to claim 7, wherein the localdispersible phase is created by means of at least one fragmentationagent which is an agent of such a nature that when combined with saidhomogeneous phase, or those constituents which will form saidhomogeneous phase, it creates said dispersible phase.
 9. The methodaccording to claim 8, wherein the fragmentation agent is selected fromthe group consisting of lipopolysaccharides, polysaccharides,glycoproteins and proteins.
 10. The method according to claim 8, whereinthe fragmentation agent is selected from the group consisting ofamphiphilic macromolecules and lipids.
 11. The method according to claim10, wherein the fragmentation agent is an amphiphilic polymer.
 12. Themethod according to claim 8, wherein the fragmentation agent is selectedfrom the following groups of amphiphilic compounds: nonionic; anionic;cationic; zwitterionic; lipids; and glycolipids.
 13. The methodaccording to claim 8, wherein the local dispersible phase is created bya procedure according to any one of the following alternatives:a) asolution of said fragmentation agent in a polar solvent is added to saidhomogeneous phase; b) a dispersion of said fragmentation agent in apolar liquid is added to said homogeneous phase; or c) said homogeneousphase is fragmentated in a solution of said fragmentation agent in apolar solvent.
 14. A method according to claim 7, wherein the particlesformed are after treated by means of one or more of the followingprocedures: homogenization, sterilization, stabilization by the additionof polymers capable of enhancing stability of colloidal systems andfreeze drying.
 15. The method according to claim 7, wherein at least onebioactive agent or a precursor thereof is added at any stage of thepreparation of said particles.
 16. Particles according to claim 1,wherein said particles are colloidal particles.
 17. Particles accordingto claim 7, wherein said particles are colloidal particles.
 18. Themethod according to claim 15, wherein said bioactive agent or aprecursor thereof is selected from the group consisting of peptides andproteins, or proteinaceous compounds.
 19. A pharmaceutical compositionof matter comprising the particles according to claim 1 and apharmaceutically acceptable carrier therefor.
 20. The pharmaceuticalcomposition of matter according to claim 1 which is in sustained releaseform.
 21. Particles prepared according to the method of claim
 7. 22. Anantigen-presenting system comprising the particles according to claim 1and a pharmaceutically acceptable carrier therefor.
 23. A colloidal drugdelivery system comprising nutrients for parenteral delivery ofnutrition and the particles according to claim
 1. 24. A method for theparenteral delivery of nutrition, said method comprising parenterallyadministering a colloidal drug delivery system comprising nutrientssuitable for the parenteral delivery of nutrition and the particlesaccording to claim 1 to a patient in need of such nutrition.
 25. Amethod for the treatment of infections, said method comprisingadministering an effective amount of an antifungal or antimicrobial drugand the particles according to claim 1 to treat an infection in apatient in need of such treatment.
 26. A method for the treatment ofcancer or AIDS, said method comprising adminstering an anticancer oranti-AIDS effective amount of a drug and the particles according toclaim 1 to treat cancer or AIDS in a patient in need of such treatment.27. A method for conducting cell culture technique or an immunoassaycomprising loading the particles according to claim 1 with a nutrientfor cells, immunomodulators, or growth factors.
 28. A method forconducting a biosensor application or using a radiation tracer, saidmethod comprising loading the particles according to claim 1 with aradiological contrast agent.
 29. The method according to claim 12,wherein said nonionic fragmentation agent is a polyethyleneoxidesurfactant.
 30. The method according to claim 12, wherein said cationicfragmentation agent is a quaternary ammonium compound.
 31. The methodaccording to claim 12, wherein said zwitterionic fragmentation agent isa phospholipid.
 32. The method according to claim 11 wherein saidamphiphilic polymer is an amphiphilic block copolymer.
 33. The methodaccording to claim 13 wherein said polar solvent is water.
 34. A methodfor conducting an immobilization technique comprising loading an enzymeinto the particles according to claim 1 as a carrier.
 35. A method forgrowing single crystals of proteins or inorganic substances, comprisingmineralizing or crystallizing pore space of the particles according toclaim 1.